Animals, Immunization and Sample Collection

Female rhesus macaques were immunized at week 0 (intranasally and orally) and week 12 (intratracheally) with replicating Ad5hr recombinants separately expressing SIV_smH4env, SIVgag, and SIVnef (n = 38, Ad-SIV) at a dose of 5 × 10⁸ plaque forming units/recombinant/site or with Ad5hr empty vector (n = 22, Ad-Empty) at a dose equivalent to the Ad-SIV recombinants administered. All rhesus macaques were maintained at the NCI animal facility under the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. The protocols and procedures were approved by the NCI Animal Care and Use Committee. Immunization and sample collection schedules are shown in Figure 1. Rectal biopsies were obtained from macaques (20 Ad-SIV immunized, 10 Ad-Empty controls) before, at day 3 and day 21 after each immunization. The biopsies were digested with collagenase (2 mg/ml, Sigma Aldrich) and single cells were harvested as described (29). Blood samples were collected before and at day 7 post-immunizations (18 Ad-SIV immunized, 12 Ad-Empty controls). PBMCs were prepared by centrifugation over Ficoll gradients as described (30). Axillary LN biopsies were collected before immunization, and inguinal LN biopsies were obtained at day 14 after the 2nd immunization (19 Ad-SIV immunized, 11 Ad-Empty controls). Biopsies were minced and passed through a 40 μm cell strainer to obtain single cells as described (20). PBMCs and LN cells were frozen in fetal bovine serum containing 10% DMSO and stored in liquid nitrogen until use. Rectal cells were used fresh immediately after the harvest.

Immunization and sample collection schedule. The immunization was given two times mucosally with a 12-weeks interval. Rectal biopsies were collected before and at day 3 and 21 post immunizations, Blood was collected before and at day 7 post immunizations. Axillary LNs were collected before the immunization and Inguinal LNs were collected at day 14 after the 2nd immunization.